Opioids remain the major medication for perioperative and chronic pain management despite their side effects, which include respiratory depression, constipation, addiction and others related to morbidity and mortality in our patients, as well as presenting a significant economic and political burden. Since both the analgesic and side effects are thought to arise from activation of the -receptor (MUR), a better understanding of the structural and functional relationships of human MUR will provide clues in addressing this clinical dilemma. In this project, we will engineer water soluble variants of the human mu receptor (wsMUR) based on the newly available crystal structure of murine MUR, investigate the properties of the receptor in the presence or absence of N and C terminus, and establish a system in investigating the direct interaction of an opioid ligand with the receptor without needing radioactive ligands or mammalian cells. There are 2 major aims for this study. In specific aim 1, we will engineer and study a new generation of wsMUR based on the crystal structure of murine MUR along with our first version of engineered receptor published recently and establish a novel system in investigating the direct interaction of opioid and receptor by taking the advantages of wsMUR and surface plasmon resonance. In specific aim 2, we will computationally engineer variants of wsMURs by changing wide ranges of surface residues in the transmembrane region to test the following hypotheses: 1) There is a minimal requirement of the number of mutations enabling the human MUR to be expressed in E coli with a reasonable water solubility and comparable properties to its native form~ 2) The receptor may tolerate 35% of the mutations for the residues in the transmembrane portion based on our past experiences of protein engineering on the membrane proteins to maximize water solubility and improve monomeric states for future high resolution protein dynamics studies (i.e. nuclear magnetic resonance) in solution conditions~ 3) Removal of cysteine on the surface of the transmembrane portion of the receptor could reduce disulfide bond induced protein dimerization. This project not only provides various variants of wsMUR for structure function relationship studies, but also yields a novel system that can investigate the direct interaction between opioids and MUR, and offer a significant technology breakthrough. Thus, this project stands to elucidate the molecular mechanisms of MUR and aid in the identification of novel opioid analgesics with reduced side effects, improved pain control, and address issues related to opioid administration and abuse. This group has a track record in engineering water soluble membrane proteins. Professor Saven at the Department of Chemistry is an expert on protein engineering and Professor Liu at the Department of Anesthesiology is an expert on opioid receptors. The preliminary data for all the specific aims are promising.